Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma

Xavier Martiáñez-Vendrell,Babacar Faye,Katherine Torres,Alfredo Mayor,Ana Vásquez,Sandra Incardona,Raquel González,Xavier C Ding,Ana Campillo,Dionicia Gamboa,Alfons Jiménez,Clara Menéndez,Wellington Oyibo,Eusebio Macete

doi:10.1186/s12936-019-3083-5

Abstract

BackgroundMalaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries.ResultsThe qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density.ConclusionThis immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

Highlights

Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase
Two MagPlex® microspheres (Luminex Corp., Austin, TX, USA) with different spectral signatures selected for the detection of PfHRP2 and PAN-Plasmodium spp lactate dehydrogenase (pLDH) were washed with distilled water and activated with Sulfo-NHS (N-hydroxysulfosuccinimide) and 1-ethyl-3-[3-dimethylaminopropyl] carbodimide hydrochloride (EDC) (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) (Pierce, Thermo Fisher Scientific Inc., Rockford, IL, USA), both at 50 mg/ mL, in activation buffer (100 mM Monobasic Sodium Phosphate, pH = 6.2)
Higher median fluores‐ cence intensity (MFI) values were obtained for PfHRP2 type A compared to types B and C, to previously reported data [24, 25]

Summary

Introduction

Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). P. falciparum infections can remain undetectable in peripheral blood as the parasites sequester in the intervillous spaces of the placenta by specific adhesion to chondroitin sulfate A [8, 9]. In such scenario, PfHRP2-detecting RDTs have been shown to have higher sensitivity on peripheral blood compared to conventional light microscopy [10], still lower than PCR [11]

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Conclusion